The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY15 we reported discoveries in the following areas: 1. atherosclerosis; 2. Alzheimer's Disese, 3. The primary immunodeficiency disease WHIM syndrome, and 4. mechanisms of allograft tolerance. 1.) In FY15, we continued our work published over the past decade on chemokine regulation of atherosclerosis. In those previous papers we described roles for CX3CR1, CCR6 and CCR7 in the ApoE knockout mouse model of atherosclerosis. In the present reporting period we found that the Atypical chemokine receptor Ackr1 (Duffy antigen receptor for chemokines) facilitated atherogenesis in the same model. Although the phenotype was robust, occurring even in littermates, and could be tracked to Ackr1 on hematopoietic cells by radiation chimera experiments, future work will be required to determine the precise molecular and cellular mechanisms. 2.) In FY15, we reported that neuronal Cx3cr1 deficiency protects against amyloid-beta-induced neurotoxicity. Previous reports had emphasized roles for Cx3cr1 in microglial cells in the brain in inflammation and in Alzheimer's models. Here we showed at the cellular level that Cx3cr1 was expressed directly on mouse neurons at both the RNA level and protein level (by immunohistochemistry). We also showed that neurons lacking Cx3cr1 produced less pro-inflammatory cytokine in response to the amyloidogenic protein amyloid-beta, a hallmark protein in Alzheimer's Disease. In collaboration with Ole Paulsen's lab in Cambridge, we extended these results to the single cell electrophysiologic level and showed that specific structural forms were required to induce neurotoxicity in a Cx3cr1-dependent manner in neurons. Together the results suggest the Cx3cr1 may be important in amyloid-beta induced neurotoxicity and support its further consideration as a potential drug target in the disease. Confirmatory studies in human are needed as well as studies comparing the quantitative importance of Cx3cr1 deficiency on microglia as compared to neurons. 3.) In FY15, we reported the case of a woman born with WHIM syndrome, a rare immunodeficiency disorder, who was cured as an adult by a second and this time fortuitous mutation that deleted the first disease-causing mutation. This remarkable case is the first report of a positive clinical result from chromothripsis, or chromosome shattering, a chromosomal catastrophe that has been associated with cancer. In addition to the disease gene, CXCR4, 163 other genes were deleted as a result of the chromothriptic event on one copy of chromosome 2 in the patient. Apparently, this occurred in one hematopoietic stem cell, since all of the patient's myeloid cells are now chromothriptic, that is, they are clonally derived from the original chromothriptic stem cell. We then showed that simple deletion of one copy of CXCR4 in a mouse, and not any of the other 163 genes that were deleted in the patient, is sufficient to give hematopoietic stem cells a strong engraftment advantage in competitive mouse transplantation experiments. This suggests a mechanism for the patient's cure (CXCR4 haploinsufficiency) and a cure strategy for other patients with WHIM syndrome using genome editing techniques that target CXCR4. CXCR4 knockdown may also be useful as an adjunct to specific gene therapy for patients with other inherited diseases of the blood. This joins a treatment strategy using the CXCR4 antagonist plerixafor in patients with WHIM syndrome (NCT00967785), which we reported in FY13 and FY14, to form a two prong approach towards benefiting our patients. In FY15, we also published evidence that plerixafor relieves panleukopenia in WHIM syndrome primarily by mobilizing multiple subsets of leukocytes to the blood from primary immune organs (bone marrow and thymus), not from secondary immune organs (lymph node, spleen) or lung. Plerixafor-mobilized neutrophils in the blood are still able to traffic to an inflammatory site, suggesting the drug treatment of our patients may be safe in this regard. Other potential side effects are still being evaluated, and the effects of the drug on other innate and adaptive immune functions remain to be established. 4.) In FY15 we also reported that pre-treatment of allogeneic bone marrow recipients with the CXCR4 antagonist AMD3100 (plerixafor) transiently enhances hematopoietic chimerism without promoting donor-specific skin allograft tolerance. Donor specific allograft tolerance is a holy grail in transplantation that, if achieved, could massively expand the number of potential organ donors and reduce the toxicity from immunosuppression associated with the procedure. It's known that establishing hematopoietic chimerism with the donor can facilitate donor-specific organ allograft tolerance. It's also thought that engraftment of stem cells requires vacant bone marrow niches. We therefore mobilized stem cells with plerixafor in the recipient hoping to enhance donor engraftment and chimerism and hence skin allograft tolerance. We observed transient enhancement of chimerism by plerixafor pre-treatment but not durable chimerism needed to promote tolerance. It's possible that our approach only supported engraftment of short term repopulating stem cells, but we don't know the precise mechanism. We have since moved on to other niche-vacating strategies.